Bone grafts including osteogenic stem cells, and methods relating to the same

ABSTRACT

Bone grafts and constructs including stem cells are provided. Example bone grafts include osteogenic stem cells seeded on a scaffold of osteoconductive cortico-cancellous chips and/or osteoinductive demineralized bone. Example constructs include extracellular matrix on a synthetic scaffold, in which the ECM is secreted from MSCs seeded onto the synthetic scaffold. Also provided are methods of making the present bone grafts and scaffolds. Further provided are methods of promoting bone healing and treating wound healing, by administering the present bone grafts and constructs to a mammal in need thereof Also provided are kits that include the present bone grafts and/or constructs, or components thereof.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.16/009,832, filed on Jun. 15, 2018 (published as U.S. Patent Pub. No.2018-0289861), which is a continuation of U.S. patent application Ser.No. 15/362,889, filed Nov. 29, 2016, now U.S. Pat. No. 10,022,474, whichis a divisional of U.S. patent application Ser. No. 14/057,806, filedOct. 18, 2013, now U.S. Pat. No. 9,539,286, which are herebyincorporated by reference in their entireties for all purposes.

TECHNICAL FIELD

The present invention relates generally to bone and wound healing usingbone grafts and constructs that include stem cells. The inventionrelates to the bone grafts and constructs themselves. The invention alsorelates to methods of making the present bone grafts and constructs andmethods of promoting bone or wound healing in a mammal by administeringthe present bone grafts and/or constructs to the mammal. The inventionfurther relates to kits that include one or more of the present bonegrafts or constructs, or components thereof.

BACKGROUND

Bone grafting is a surgical procedure that replaces missing bone and/orrepairs bone fractures. Bone generally has the ability to regeneratewell but may require a scaffold to do so. Bone grafts may be allograft(cadaveric bone e.g., from a bone bank), autologous (i.e., boneharvested from the patient's own body, for example from the iliaccrest), or synthetic. Most bone grafts are expected to be resorbed andreplaced as the natural bone heals over time.

Successful bone grafts may include osteoconduction (guiding thereparative growth of the natural bone), osteoinduction (encouragingundifferentiated cells to become active osteoblasts), and osteogenesis(living bone cells in the graft material contributing to boneremodeling). Bone grafts are osteogenic if they contain viable cellsthat are capable of bone regeneration, which is advantageous for bonehealing. Osteogenesis occurs with autografts. Autografts are consideredosteogenic because they contain a high number of bone forming cells.However, autographs have certain drawbacks in that there is limitedavailability of autographs, and autographs may result in donor sitemorbidity.

SUMMARY

There is a need in the art for bone grafts and constructs that areosteogenic, osteoinductive and/or osteoconductive without the drawbacksof present autograph products.

The present invention is related to the development of suitable bonegrafts or constructs that contain stem cells on a scaffold. In additionto stem cells, they may include for example, a mix of osteoinductivedemineralized bone and osteoconductive cortico-cancellous chips, topromote bone healing. Examples of the present invention provide bonegrafts that are osteogenic, osteoinductive and osteoconductive. Inaddition, according to non-limiting example embodiments, the osteogeniccells may be isolated from bone marrow harvested from the iliac crest,or they may be isolated from adipose tissue, or from long bones such asfemur, tibia, humerus, etc. According to example embodiments, a finalproduct bone graft product may include DBM (demineralized bone),osteogenic stem cells, and cortico-cancellous chips.

The present invention also relates to constructs that include stem cellsseeded on a synthetic scaffold. The stem cells are then instructed usingchemical cues to undergo osteogenic differentiation. This causes theMSCs to secret extracellular matrix (ECM). The extracellular matrix(ECM) plays an important role in regulating cell behavior. Adecellularized tissue matrix provides a complex ECM-based scaffold,similar to native tissue. Current products provide a decellularizedmatrix that is derived from skin, and use this as a scaffold for cellattachment and is used to promote wound healing. However, having ECM onsynthetic scaffolds helps combine the properties of the syntheticscaffold and the unique properties of the ECM, and together helpmodulate the healing process. Having the ECM on a suitable syntheticscaffold provides better mechanical properties in addition to providingthe cues similar to that of native tissue.

The present inventors have developed unique biomaterials that combinethe properties of the scaffold and an osteoinductive and osteoconductivecell derived ECM that directs cell fate and drives bone healing.

Also provided are methods of making the present bone grafts andconstructs and methods of promoting bone and wound healing by using suchbone grafts and/or constructs.

Further provided are kits that include one or more of the present bonegrafts and/or constructs, as well as kits that include components formaking the same.

DETAILED DESCRIPTION

The present invention relates generally to bone grafts and constructsthat include stem cells. The invention also relates to methods of makingthe present bone grafts and constructs and methods of promoting bone orwound healing in a mammal by administering the present bone graftsand/or constructs to the mammal. The invention further relates to kitsthat include such bone grafts and/or constructs, or components thereof.

Additional aspects, advantages and/or other features of exampleembodiments of the invention will become apparent in view of thefollowing detailed description. It should be apparent to those skilledin the art that the described embodiments provided herein are merelyexemplary and illustrative and not limiting. Numerous embodiments ofmodifications thereof are contemplated as falling within the scope ofthis disclosure and equivalents thereto.

In describing example embodiments, specific terminology is employed forthe sake of clarity. However, the embodiments are not intended to belimited to this specific terminology. Unless otherwise noted, technicalterms are used according to conventional usage.

As used herein, “a” or “an” may mean one or more. As used herein“another” may mean at least a second or more. As used herein, unlessotherwise required by context, singular terms include pluralities andplural terms include the singular.

As used herein, “extracellular matrix,” or “ECM” is the extracellularcomponent consisting of an intricate network of proteins andpolysaccharides that are secreted by the cells and play an importantrole in cell-cell signaling. Mesenchymal Stem Cells (MSCs) generate ECMsin vivo as they differentiate in different cell types. However, MSCs maybe made to secrete the ECM ex vivo, by providing chemical cues thatwould facilitate differentiation of the MSCs.

As indicated herein, bone grafts are provided herein that include ascaffold that includes at least one of osteoinductive demineralized boneand/or osteoconductive cortico-cancellous chips; and osteogenic stemcells. According to more specific non-limiting example embodiments, bonegrafts are provided that include osteogenic stem cells in a mix ofosteoinductive demineralized bone and osteoconductive cortico-cancellouschips to promote bone healing.

According to non-limiting example embodiments, the present bone graftsmay be made by methods that include seeding osteogenic cells onto atissue culture substrate, such as a flask and allowing the cells toattach; washing the substrate to remove blood cells; detachingosteogenic stem cells from the substrate; and seeding the osteogenicstem cells on a scaffold comprising at least one of osteoinductivedemineralized bone and osteoconductive cortico-cancellous chips.

According to non-limiting example embodiments, the osteogenic stem cellsmay be obtained by treating cancellous bone from e.g., a femur or tibiawith collagenase and separating loosely attached cells bycentrifugation.

The cortico-cancellous chips may be obtained for example, from thecondyles from fresh frozen bone. In particular, the condyles may beseparated from the cortical shaft, and sectioned into smaller pieces.The decellularized condyles may be milled to form cortico-cancellouschips.

The cortical bone may be milled and demineralized in HCl to formdemineralized bone (DBM). As used herein, the terms “demineralizedbone”, “demineralized bone matrix”, and “DBM” are used interchangeablyherein.

Thus, according to example embodiments, cortical bone may be treated andprocessed to form DBM. The DBM and/or cortico cancellous chips (which beobtained for example from condyles), may be used individually or as amix or other combination together to form the scaffold.

The bone marrow from the cortical shaft may be flushed and mixed withthe centrifuged stem cells (e.g., obtained from cancellous bone). Themixture of cells may be seeded onto a tissue culture flask or othertissue culture substrate and allowed to attach thereto.

The substrate may then be washed to remove any blood cells, and the boneforming cells will be detached e.g., using trypsin. These cells willthen be seeded on the scaffold of cortico-cancellous chips,demineralized bone, or a mix or other combination of the same.

According to alternative embodiments, the isolated osteogenic stem cells(obtained e.g. after treating the cancellous bone from the femur ortibia with collagenase), may be seeded directly on a scaffold includingDBM, CC, or a mix or other combination of the same.

Thus, provided herein are methods of making bone grafts that includeseeding osteogenic cells on a scaffold; which scaffold includesosteoinductive demineralized bone and/or osteoconductivecortico-cancellous chips. According to non-limiting example embodiments,the present methods may further include rinsing the scaffold inphosphate buffered saline (PBS) to remove unwanted cells, preferably allunwanted cells, or as many as possible.

In accordance with any of these embodiments, the osteogenic cells mayinclude e.g., osteogenic cells isolated from bone marrow harvested fromiliac crest, or from long bones such as femur, tibia, humerus etc. Thus,example embodiments may include bone grafts that include osteogeniccells isolated from bone marrow harvested from iliac crest, or from longbones such as femur, tibia, humerus etc. which are seeded directly onthe scaffold of CC, DBM, or a mixture thereof. Following cellattachment, the scaffolds may be rinsed e.g., with PBS to removeunwanted cells. Thus, the stem cells in these embodiments may be fromthe same donor from which the DBM and CC are derived.

According to other non-limiting example embodiments, the osteogeniccells may include e.g., osteogenic cells isolated from adipose tissue.The adipose tissue may be sectioned and treated with collagenase enzyme.The tissue may then be either centrifuged to facilitate cell separationor incubated at 37° C. in a petri dish to facilitate cell migration fromthe tissue onto the petri dish. Thus, example embodiments may includebone grafts that include osteogenic cells isolated from adipose tissue,which are seeded directly on the scaffold of CC, DBM, or a mixturethereof. Following cell attachment, the scaffolds may be rinsed toremove unwanted cells.

According to non-limiting example embodiments, the scaffold is a mix ofdemineralized bone matrix and cortico-cancellous chips in a ratio ofabout 1:1-1:3, or any point there-between, including for example, ratiosof about 1:1, 1:2 or 1:3.

According to further example embodiments, the osteogenic stem cells maybe present in the bone graft in an amount of at least 20,000 cells/cc oftotal bone graft. The bone graft may be for example, in a semi-solidstate, such as in a putty-like state.

In any of the above embodiments, the final products may be stored at−80° C. or −180° C.

Further provided herein are methods of promoting bone healing, whichinclude administering to a mammal in need thereof, a bone graft thatincludes osteogenic stem cells and a scaffold that includesosteoinductive demineralized bone and/or osteoconductivecortico-cancellous chips.

Also provided herein, according to non-limiting example embodiments, areconstructs that include ECM on synthetic scaffolds. In theseembodiments, Mesenchymal stem cells (MSCs) are seeded onto a syntheticscaffold to form the construct. The stem cells are then instructed usingchemical cues to undergo osteogenic differentiation. This causes theMSCs to secrete the extracellular matrix onto synthetic scaffolds. Asused herein, the terms “Mesenchymal stem cells” and “MSC” are usedinterchangeably. According to non-limiting example embodiments, the MSCsare isolated from adipose tissue, bone marrow or bone. According toexample embodiments, the mesenchymal stem cells (MSCs) are derived frombone marrow or cancellous bone. The MSCs may be human or bovine derived.The MSCs in these construct embodiments may be purchased.

According to non-limiting example embodiments, the synthetic scaffoldmay include natural or synthetic materials, such as at least one ofcollagen-ceramic, collagen-bioglass, or PLGA. By way of non-limitingexample embodiment, the scaffolds may include collagen-ceramic,collagen-bioglass, or PLGA-based scaffolds. Other suitable biocompatiblescaffolds are also contemplated however.

According to non-limiting example embodiments, the present constructsmay have a seeding density of from 20,000 to 100,000 cells/cm² of thegrowth surface area on which the cells are seeded.

According to non-limiting example embodiments, the mesenchymal stemcells in the construct are cultured in media; and the construct isrinsed in PBS, treated with DNAse, and air dried.

Also included herein are methods of making constructs that includeseeding mesenchymal stem cells (MSCs) on a synthetic scaffold to form aconstruct; culturing the MSCs in supplemented media; rinsing theconstruct with PBS and treating with DNAse; rinsing the construct withPBS to remove prior reagents; and air drying the construct.

The culturing may include for example, culturing for 5-15 days insupplemented media, which may be for example, α-MEM, 10% fetal bovineserum, 1% penicillin-streptomycin, 50 μg/ml ascorbic 2 phosphate). Theculture media may be changed every 2-3 days with fresh media.

According to example embodiments, at the end of the culture period theconstruct is rinsed with PBS. After such rinsing, the construct may betreated with PBS containing 0.1-2% tritonX-100 and 10-40 mM NH₄OH for5-30 minutes, or according to other example embodiments for 5-10minutes, before further rinsing with PBS and treatment with DNAase.According to further example embodiments, the construct is treated withPBS containing 0.3-1% tritonX-100 and 10-30 mM NH₄OH. According tofurther non-limiting example embodiments, the construct is treated withPBS containing 0.5% tritonX-100 and 20 mM NH₄OH.

According to other example embodiments, at the end of the culture periodthe construct is rinsed with PBS; and thereafter, the construct may befurther processed through 2-3 freeze thaw cycles before further rinsingwith PBS and treatment with DNAase.

The treatment of the construct with DNAse may include treatment ofDNAase in a concentration of 100-300 units/ml, or of approximately 150units/ml, for approximately (0-60 min) at about 37° C., e.g., understandard tissue culture condition. Treatment with DNAse may be followedby 2-3 rinses in PBS for removal of prior reagents, preferably of allprior reagents.

According to example embodiments, the construct may then be air driedand stored on the shelf.

Also provided herein are methods of making a construct comprisingseeding mesenchymal stem cells (MSCs) onto a synthetic scaffold;culturing the cells in cell culture media; and replacing the cellculture media with freezing media that contains DMSO (e.g., about 10%),and freezing. In these embodiments, the constructs may be seeded withallogenic mesenchymal stem cells isolated from humans. The seedingdensity may be 20,000-100,000 cells/cm². The cell culture media may bereplaced e.g., after about 24 hours of attachment. The cell-basedconstructs may be stored for example at about −80° C. (e.g., aboutnegative 78° C. to −82° C.) or vapour phase liquid nitrogen.

The constructs may be for example, in solid or putty form.

Further provided are methods of treatment for wound healing that includeadministering to a mammal in need thereof any of the present constructs,including e.g., ECM.

Non-limiting example embodiments also include kits that include one ormore of the present bone grafts and/or constructs and optionallyinstructions for preparing such constructs or bone grafts and/or forusing them for bone healing or wound healing.

Further example embodiments are directed to kits that include componentsfor making the present bone grafts and/or constructs, including forexample, synthetic scaffolds, cell culture media, PBS,cortico-cancellous chips, demineralized bone, a tissue culture substratesuch as a flask, trypsin, or mixtures or other combinations thereof.Additional components, instructions and/or apparatus' may also beincluded.

The following examples are provided to further illustrate variousnon-limiting embodiments and techniques. It should be understood,however, that these examples are meant to be illustrative and do notlimit the scope of the claims. As would be apparent to skilled artisans,many variations and modifications are intended to be encompassed withinthe spirit and scope of the invention.

EXPERIMENTAL EXAMPLES Example 1

In this example, a bone graft is formed in accordance with the presentinvention. Condyles from fresh frozen bone may be separated from thecortical shaft, and sectioned into smaller pieces. The osteogenic cellsin this example may be obtained after treating the cancellous bone fromthe femur or tibia with collagenase. The loosely attached cells may thenbe separated by centrifugation. The decellularized condyles may bemilled to form cortico-cancellous chips (CC). The bone marrow from thecortical shaft may be flushed and mixed in with the centrifuged cells.

The mixture of bone marrow and osteogenic cells may be seeded onto atissue culture substrate such as a flask and allowed to attach. Thesubstrate may be washed to remove any blood cells, and the bone formingcells detached using trypsin.

The cortical bone may be milled and demineralized e.g., in HCl to formDBM. The DBM may then be mixed with cortico-cancellous chips. The cellsmay then be seeded on the scaffold which could be CC, DBM, or a mix orother combination of CC and DBM to form a bone graft.

Example 2

In this example, the osteogenic cells (isolated as set forth inExample 1) may be seeded directly on the scaffold, which could be DBM,CC, or a mix of DBM and CC. After osteogenic cell attachment, thescaffolds may be rinsed in phosphate buffered saline (PBS) to remove allunwanted cells.

Example 3

In this example, the osteogenic cells are isolated from bone marrowharvested from the iliac crest. The harvested bone marrow may then beseeded directly on the scaffold (CC, DBM, or a mix of CC and DBM).Following cell attachment, the scaffolds may be rinsed to removeunwanted cells.

Example 4

In this example, the osteogenic cells are isolated from adipose tissue.The adipose tissue may be sectioned and treated with collagenase enzyme.The tissue may then be either centrifuged to facilitate cell separationor incubated at 37° C. in a petri dish to facilitate cell migration fromthe tissue onto the petri dish. The isolated cells may then be seeded onthe scaffold (e.g., CC chips, DBM, or mix or other combination of CCchips and DBM).

Example 5

In this example, a construct is prepared in accordance with embodimentsof the present composition is prepared. Mesenchymal stem cells (MSCs)either derived from bone marrow or cancellous bone are seeded ontocollagen-ceramic scaffold. The MSCs may be human or bovine derived. Theseeding density may be 20,000-100,000 cells/cm². The cells are culturedfor 5-15 days in supplemental media (α-MEM, 10% fetal bovine serum, 1%penicillin-streptomycin, 50 μg/ml ascorbic 2 phosphate). The culturemedia is changed every 2-3 days with fresh media. At the end of theculture period, the construct may be rinsed with PBS and treated withPBS containing 0.5% triton-100 and 20 mM NH₄OH, for 5-10 min. At the endof the treatment the constructs are rinsed in PBS and treated with DNAse(150 units/ml) for 1 hour at 37° C. This treatment will be followed by2-3 rinses in PBS to confirm complete removal of all prior reagents. Theconstruct may then be air dried and stored on the shelf.

Example 6

In this example, Mesenchymal stem cells (MSCs) either derived from bonemarrow or cancellous bone are seeded onto a collagen-ceramic scaffold.The MSCs may be human or bovine derived. The seeding density may be20,000-100,000 cells/cm². The cells are cultured for 5-15 days insupplemented media (α-MEM, 10% fetal bovine serum, 1%penicillin-streptomycin, 50 μg/ml ascorbic 2 phosphate). The culturemedia may be changed every 2-3 days with fresh media. At the end of theculture period, the construct is rinsed with PBS and processed through2-3 freeze thaw cycles. A freeze thaw cycle includes freezing constructsin liquid nitrogen for 3-5 min, thawing at 37° C. water bath for 5 min,and rinsing constructs in PBS. At the end of the treatment theconstructs are rinsed in PBS and treated with DNAse (150 units/ml) for 1hr at 37° C. This treatment will be followed by 2-3 rinses in PBS toconfirm removal of all prior reagents. The construct may then be airdried and stored on the shelf.

Example 7

In this example, the constructs from Examples 5 and 6 may be seeded withallogenic mesenchymal stem cells isolated from humans. The seedingdensity may be 20,000-100,000 cells/cm². After 24 hr of attachment thecell culture media may be replaced with freezing media that contains 10%DMSO, and frozen. The cell based constructs may be stored at −80 ° C. orvapour phase liquid nitrogen.

Although the invention has been described in example embodiments, thoseskilled in the art will appreciate that various modifications may bemade without departing from the spirit and scope of the invention. It istherefore to be understood that the inventions herein may be practicedother than as specifically described. Thus, the present embodimentsshould be considered in all respects as illustrative and notrestrictive. Accordingly, it is intended that such changes andmodifications fall within the scope of the present invention as definedby the claims appended hereto.

What is claimed is:
 1. A composition having viable cells comprising: aputty-like bone graft stored in a freezing media comprising DMSO underfreezing condition, the putty-like bone graft comprising osteogenic stemcells and a scaffold, the scaffold comprising a mixture ofcortico-cancellous chips and demineralized cortical bone, thecortico-cancellous chips containing bone material from both cancellousbone and cortical bone contained in condyles and only the condylesexcluding the cortical shaft, the demineralized cortical bone containingbone material in cortical shafts of long bones, wherein the osteogenicstem cells are viable cells capable of bone regeneration.
 2. Thecomposition of claim 1, wherein the osteogenic stem cells are present inan amount of 20,000 to 100,000 cells/cm².
 3. The composition of claim 1,wherein the osteogenic stem cells are mesenchymal stem cells.
 4. Thecomposition of claim 3, wherein the mesenchymal stem cells are derivedfrom a human source.
 5. The composition of claim 3, wherein themesenchymal stem cells are derived from a bovine source.
 6. Thecomposition of claim 1, wherein the osteogenic stem cells, demineralizedcortical bone, and cortico-cancellous chips are derived from the samedonor.
 7. The composition of claim 1, wherein the demineralized corticalbone and cortico-cancellous chips are present in a ratio of about 1:1demineralized cortical bone to cortico-cancellous chips.
 8. Thecomposition of claim 1, wherein the demineralized cortical bone andcortico-cancellous chips are present in a ratio of about 1:2demineralized cortical bone to cortico-cancellous chips.
 9. Thecomposition of claim 1, wherein the demineralized cortical bone andcortico-cancellous chips are present in a ratio of about 1:3demineralized cortical bone to cortico-cancellous chips.
 10. Acomposition having viable cells comprising: a putty-like bone graftstored in a freezing media comprising DMSO under freezing condition, theputty-like bone graft comprising osteogenic stem cells and a scaffold,the scaffold comprising a mixture of non-demineralizedcortico-cancellous chips and demineralized cortical bone which form thescaffold without an additional natural or synthetic material, thenon-demineralized cortico-cancellous chips containing bone material fromboth cancellous bone and cortical bone contained in condyles and onlythe condyles excluding the cortical shaft, the demineralized corticalbone containing bone material in cortical shafts of long bones whereinthe osteogenic stem cells are viable cells capable of bone regeneration.11. The composition of claim 10, wherein the osteogenic stem cells arepresent in an amount of 20,000 to 100,000 cells/cm².
 12. The compositionof claim 10, wherein the osteogenic stem cells are mesenchymal stemcells.
 13. The composition of claim 12, wherein the mesenchymal stemcells are derived from a human source.
 14. The composition of claim 12,wherein the mesenchymal stem cells are derived from a bovine source. 15.The composition of claim 10, wherein the osteogenic stem cells,demineralized cortical bone, and non-demineralized cortico-cancellouschips are derived from the same donor.
 16. The composition of claim 10,wherein the demineralized cortical bone and non-demineralizedcortico-cancellous chips are present in a ratio of about 1:1demineralized cortical bone to cortico-cancellous chips.
 17. Thecomposition of claim 10, wherein the demineralized cortical bone andnon-demineralized cortico-cancellous chips are present in a ratio ofabout 1:2 demineralized cortical bone to cortico-cancellous chips. 18.The composition of claim 10, wherein the demineralized cortical bone andnon-demineralized cortico-cancellous chips are present in a ratio ofabout 1:3 demineralized cortical bone to cortico-cancellous chips.